Compositions and method for accurate early pregnancy diagnosis

ABSTRACT

The invention provides improved assays for detection of pregnancy. In the assays, pregnancy associated glycoproteins are analyzed in conjunction with progesterone analysis. The techniques of the invention overcome limitations in the prior art by reducing the rate of false positive results. The assays provided by the invention can be implemented to increase the efficiency of commercial animal breeding programs.

The present application claims benefit of priority from U.S. ProvisionalSer. No. 60/331,822, filed Nov. 20, 2001, the entire contents of whichare hereby incorporated by reference.

BACKGROUND OF THE INVENTION

I. Field of the Invention

The present invention relates generally to the fields of veterinarymedicine, reproductive biology and diagnostics. More specifically, thepresent invention relates to improved methods for early stage pregnancydetection.

II. Related Art

Pregnancy diagnosis is an important component in sound reproductivemanagement, particularly in the dairy industry (Oltenacu et al., 1990),where a high proportion of artificial inseminations fail (Streenan andDiskin, 1986). A reliable yet simple pregnancy test for cattle has longbeen sought. Several procedures are available, including a milkprogesterone assay (Oltenacu et al., 1990; Markusfeld et al., 1990),estrone sulfate analysis (Holdsworth et al., 1982; Warnick et al.,1995), rectal palpation (Hatzidakis et al., 1993), ultrasound (Beal etal., 1992; Cameron and Mahno, 1993), and blood tests forpregnancy-specific antigens. Of these, the progesterone milk assay isthe most cost effective for the producer (Oltenacu et al., 1990;Markusfeld et al., 1990). Next best is rectal palpation, performed atday 50 post-insemination (Oltenacu et al., 1990).

Even though the prior procedures for pregnancy diagnosis are potentiallyuseful, all have fallen short of expectations in terms of theirpractical, on-farm use. For example, measurements of milk or serumprogesterone around day 18-22 yield unacceptably high rates of falsepositives (Oltenacu et al., 1990; Markusfeld et al., 1990). The presenceof estrone sulfate in urine or serum provides another test, but is onlyuseful after day 100 as concentrations rise (Holdsworth et al., 1982;Warnick et al., 1995).

The discovery of pregnancy-specific protein B (PSP-B) (Butler et al.,1982) provided a new approach to pregnancy diagnosis since it could bedetected in the blood of pregnant cows by the fourth week of pregnancy(Sasser et al., 1986; Humblot et al., 1988). Two other groups havedeveloped immunoassays that may be based on an identical orimmunologically similar antigen (Zoli et al., 1992a; Mialon et al.,1993; Mialon et al.., 1994). In one case, the antigen (Mr ˜67 kDa) wascalled bovine pregnancy-associated glycoprotein (boPAG; now boPAG-1)(Zoli et al., 1992a); in the second, it was designated as pregnancyserum protein 60 (PSP60) (Mialon et al., 1993; Mialon et al., 1994). Theimmunoassay for PSP-B/boPAG1/PSP60 has two advantages. First, it candetect pregnancy relatively early. Second, interpretation of the assaysdoes not require knowledge of the exact date of service, since boPAG-1immunoreactive molecules are always present in the maternal serum ofpregnant cows by day 28, and concentrations increase as pregnancyadvances (Sasser et al., 1986; Mialon et al., 1993; Mialon et al.,1994).

There remain, however, two major disadvantages to this procedure. First,positive diagnosis in the fourth week of pregnancy remains somewhatuncertain because antigen concentrations in blood are low and somewhatvariable. Second, boPAG1 concentrations rise markedly at term (Sasser etal., 1986; Zoli et al., 1992a; Mialon et al., 1993) and, due to the longcirculating half-life of the molecule (Kiracofe et al., 1993), theantigen can still be detected 80-100 day postpartum (Zoli et al., 1992a;Mialon et al., 1993; Mialon et al., 1994; Kiracofe et al., 1993),compromising pregnancy diagnosis in cows bred within the earlypostpartum period. Thus, the test can be carried out in dairy cows atday 30 only if artificial insemination (“AI”) is performed at 45-70 dayspost-partum.

Analysis of other BoPAGs in particular has exhibited potential for usein pregnancy testing. However, such tests can yield high false positiverates. This error rate occurs because the PAG test is done at day 25 ofpregnancy. However, some embryos die between day 20 and 30 of pregnancy.This dying tissue can probably produce some PAG. Thus, the cow is PAGpositive, but the embryo is dead. The results of this can be a falsepositive rate of 8%, which is generally considered to be unacceptablewithin commercial breeding programs. There is, therefore, a need forpregnancy tests with improved accuracy.

A pregnancy test that could be carried out reliably and early inpregnancy could provide definitive indication as to whether rebreedingor culling is required. In general, AI is successful less than 50% ofthe time and the producer must either rely on overt signs of return toestrus (that are easily missed) or delay rebreeding until pregnancyfailure is confirmed by one of the methods described above. Such delaysare extremely costly and constitute a major economic loss to theindustry. There is thus a need for a feasible, sensitive and accuratepregnancy test in cattle that yields a low level of false positiveresults.

SUMMARY OF THE INVENTION

The invention provides methods for the early detection of pregnancy inlivestock such as ungulates (e.g., hoofed animals). In one aspect of theinvention, methods are provided for the early detection of pregnancy ina bovine animal comprising: (a) obtaining a sample from the bovineanimal; (b) measuring the level of at least one bovine pregnancyassociated antigen (BoPAG) in the sample; and (c) measuring the level ofprogesterone in the sample, wherein elevated levels of BoPAG andprogesterone indicate that the bovine animal is pregnant. The sample maybe from any biological material, including saliva, serum, blood, milk orurine. In certain embodiments of the invention, the sample may beobtained from the animal at days 16 to 30, days 16 to 28, days 16 to 25and days 20 to 25 post-insemination, including about day 20, 25, 28 or30 post-insemination. The analysis may comprise measuring the level ofmore that one BoPAG and, in certain embodiments, may comprise measuringone or more BoPAGs selected from the group consisting of BoPAG1, BoPAG2,BoPAG3, BoPAG4, BoPAG5, BoPAG6, BoPAG7, BoPAG8, BoPAG9, BoPAG7v;BoPAG9v; BoPAG10, BoPAG11, BoPAG12, BoPAG13, BoPAG14, BoPAG15; BoPAG16;BoPAG17; BoPAG18; BoPAGl9; BoPAG20 or BoPAG21, including anycombinations thereof The BoPAG may also be present in early pregnancyand may, for example, be selected from the group consisting of BoPAG2,BoPAG4, BoPAG5, BoPAG6, BoPAG7, BoPAG8, BoPAG9, BoPAG10, BoPAG11 andBoPAG21. Alternatively, the BoPAG may be present throughout pregnancy,and may also, for example, be selected from the group consisting ofBoPAG2, BoPAG8, BoPAG10 and BoPAG11.

In one embodiment of the invention, a BoPAG that is analyzed is presentin early pregnancy and absent at about two months post-parum. The BoPAGmay, for example, be selected from the group consisting of BoPAG2,BoPAG4, BoPAG5, BoPAG6, BoPAG7, and BoPAG9. The measuring may compriseimmunologic detection, including detecting a plurality of BoPAGs withpolyclonal antisera. The polyclonal antisera may lack substantialbinding activity to BoPAG1. In another embodiment of the invention, thepolyclonal antisera is prepared against acidic fraction of day 75-85BoPAG or comprises detecting a single BoPAG with a monoclonal antibodypreparation. The immunologic detection may also comprise detection ofmultiple BoPAGs with a monoclonal antibody preparation. Immunologicdetection may be carried out using any technique, including ELISA, RIA,and Western blot. The ELISA may comprise a sandwich ELISA comprisingbinding of a BoPAG to a first antibody preparation fixed to a substrateand a second antibody preparation labeled with an enzyme. In oneembodiment, the enzyme is alkaline phosphatase or horseradishperoxidase. In certain embodiments of the invention, an elevated levelof total BoPAG that is detected is from about 5 to about 10 ng/ml ofserum, including about 5 ng/ml and 10 ng/ml. Measuring BoPAG levels maycomprise, for example, nucleic acid hybridization, including Northernblotting and nucleic acid hybridization comprises amplification. Theamplification may comprise RT-PCR.

In the method, measuring progesterone levels may also compriseimmunologic detection. In certain embodiments of the invention,immunologic detection may comprise detecting progesterone withpolyclonal antisera or detecting progesterone with a monoclonal antibodypreparation. Immunologic detection may be carried out using anytechnique, including ELISA, RIA, and Western blot. The ELISA maycomprise a sandwich ELISA comprising binding of a progesterone to afirst antibody preparation fixed to a substrate and a second antibodypreparation labeled with an enzyme. In one embodiment, the enzyme isalkaline phosphatase or horseradish peroxidase. The elevated level ofprogesterone that is detected may, in certain embodiments of theinvention, comprise about 2 ng/ml of serum.

In certain embodiments of the invention, a sample is obtained at aboutday 25 post-insemination, and the elevated levels of BoPAG andprogesterone are about 10 ng/ml and 2 ng/ml, respectively. A positivecontrol sample may also be obtained from a pregnant bovine animal, asmay a negative control sample from a non-pregnant bovine animal. Themethod may further comprise measuring BoPAG and progesterone levels froma second sample from the bovine animal at a second point in time.

In another aspect, the invention provides a method of making a breedingdecision for a bovine animal comprising: (a) obtaining a sample from thebovine animal, wherein the bovine animal is suspected of being pregnant;(b) measuring the level of at least one bovine pregnancy associatedantigen (BoPAG) in the sample; and (c) measuring the level ofprogesterone in the sample, wherein: (i) elevated levels of BoPAG andprogesterone indicate that the bovine animal is pregnant, and no furthersteps need be taken; (ii) non-elevated levels of BoPAG and progesteroneindicate that the bovine animal is not pregnant, and should be injectedwith gonadotropin-releasing hormone (GnRH), and about seven days later,injected with prostaglandin F_(2α) (PGF), followed by re-insemination;(iii) elevated levels of BoPAG and non-elevated levels of progesteroneindicate that the bovine animal is not pregnant due to early embryodeath and should be injected with GnRH, and about seven days later,injected with PGF, followed by re-insemination; or (iv) non-elevatedlevels of BoPAG and elevated levels of progesterone indicate that thebovine animal is not pregnant, and should be injected with PGF, followedby re-insemination. The method may also further comprise steps (ii),(iii) and (iv), about 48 hours after PGF injection and beforere-insemination, administering a second injection of GnRH. The methodmay also further comprise, prior to step (a), inseminating the bovineanimal.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The invention overcomes the limitations of the prior art by providing areliable test for early pregnancy diagnosis and methods for use thereof.A reliable yet simple pregnancy test for cattle has long been sought.Typical prior test have either not allowed early detection of pregnancyor have suffered from a high incidence of false positive or falsenegative results. The prior tests, although potentially useful, havethus fallen short of expectations in terms of their practical, on-farmuse.

The present invention overcomes the limitations of the prior art byanalyzing placentally expressed polypeptides, designated pregnancyassociated glycoproteins (PAGs), in conjunction with progesterone levelsfor the early and accurate diagnoses of bovine and other pregnancies. Inparticular, the inventors have found that by assaying for bothprogesterone and PAGs, early pregnancy diagnosis is possible with a highdegree of accuracy. This is because the combined test measures a fetalcomponent (PAG) and a maternal component (progesterone), both of whichare essential for the establishment of successful pregnancy in cattleand other livestock species. The finding is significant becausepregnancy diagnosis is an important component in reproductive managementof livestock, particularly in the dairy industry where a high proportionof artificial inseminations fail and additional days open reduce the netoperating income to the producer.

The tests of the invention can be carried out by detection of PAG andprogesterone in the serum of animals, including bovines, in earlypregnancy. In one embodiment of the invention, the assay can be carriedout using polyclonal antibodies raised against early PAG enrichedfraction. For example, such a fraction was purified by the inventorsfrom day 80 bovine placenta. Alternatively, individual PAGs orcombinations of PAGs can be analyzed as is described herein below.Methods for carrying out analysis of PAGs are disclosed in U.S. patentapplication Ser. No. 09/273,164, filed Mar. 19, 1999, the entiredisclosure of which is specifically incorporated herein by reference.For progesterone analysis, commercially available assay kits areavailable that may be used to measure serum levels of progesterone.Using a PAG assay and the commercial progesterone assay, it was foundthat pregnancy detection could be performed as early as day 25 and withvery low (<5%) false positive and false negative results.

I. LIVESTOCK BREEDING PROGRAMS

One advance of the current invention is that it allows early detectionof pregnancy with a low incidence of false positive results. Earlydetection of pregnancy is important because it allows rebreeding ofanimals found to not be pregnant. A low incidence of false positives isnecessary to allow implementation of an effective rebreeding protocol.Prior pregnancy tests typically either were unable to be used for earlytesting or exhibited high incidence of false positives.

A type of early pregnancy test which has been used is the detection ofpregnancy associated antigens (PAGs). An advantage of this test is thatit can be done at day 25 of pregnancy. However, some embryos die betweenday 20 and 30 of pregnancy and, in some cases, the dying tissue mayproduce PAG. Thus cows may be PAG positive but the embryo is dead.

As discussed herein below, the inventors have found that by analyzingprogesterone levels in addition to PAGs, a very low incidence (<5%) offalse positives can be obtained. This is because the corpus luteumregresses shortly after embryo death. Thus, a cow with a dying embryowould have PAG but low progesterone. Because the progesterone is anabsolute requirement for establishing pregnancy, a cow with low serumprogesterone cannot maintain pregnancy.

A. Estrus and ovulation

Dairy cows come into estrus once every 21 days. Cows displaycharacteristic behaviors during estrus. Farmers can identify cows inestrus by these characteristic behaviors. Cows ovulate an egg about 28hours after the onset of estrus. Most dairy cows are inseminatedartificially about 12 hours after the onset of estrus so that sperm arein the reproductive tract when the cow ovulates.

B. Efficiency of reproduction in dairy cows

Lactating dairy cows are watched for estrus. They are inseminated whenthey come into estrus so that they can become pregnant and have anothercalf. The efficiency with which cows are detected in estrus is low. Onlyabout 50% of cows in estrus are actually detected by farmers. Of thecows detected in estrus and inseminated, only about 30% will becomepregnant. Thus, only about 15% (50%×30%) of ovulations result in apregnancy. Dairy reproduction is inefficient because cows in estrus arenot always seen and those inseminated don't always get pregnant.Although most cows could be inseminated once every 21 days (assumingthey do not get pregnant), the true insemination interval on farms isonce every 40 to 60 days. The lost time results in frustration becausefarmers want their cows pregnant as soon as possible. There are alsoeconomic implications to the delay. The efficiency of reproduction hasworsened since 1985 because of consolidation of the dairy industry(larger farms, less human-cow contact, labor shortages, etc.). Dairymenare very concerned about reproduction. Most dairy cows are culledbecause they do not get pregnant.

C. Corpus luteum and progesterone After a cow ovulates a corpus luteum(CL) is formed on the ovary that secretes a hormone called progesterone.Progesterone can be detected in the blood of the mother. Progesterone isneeded to maintain the pregnancy. If the egg is fertilized and theembryo grows and survives then the corpus luteum will be maintaineduntil the end of gestation (280 days). If the egg is not fertilized orthe embryo dies then the corpus luteum will regress. The cow comes backinto estrus after the corpus luteum regresses and can be inseminatedagain if seen in estrus..

D. PAG and pregnancy testing

The developing embryo produces PAGs. These can be detected in the bloodof the mother at about 25 days of pregnancy. The PAG pregnancy test isdesigned to detect PAGs in the blood of the mother. A pregnant cow willalso have high progesterone in blood because she will have a corpusluteum. Thus, pregnant cows will have PAG and progesterone in blood.

E. Pregnancy testing in dairy

The problem with reproductive management in dairy cattle is thatpregnancy detection has previously typically been done 35 to 60 daysafter breeding. Furthermore, most nonpregnant cows are simply injectedwith PGF because the status of the corpus luteum is not known. However,the pregnancy tests of the current invention can be done 10 to 35 dayssooner than these traditional pregnancy testing and only cows with a CLcan be injected with PGF. Cows that do not have a CL (and will notrespond to PGF) can be injected with GnRH and then treated with PGF atthe appropriate time. By implementing this plan, farmers will know whichcows are pregnant and also inseminate nonpregnant cows within about 30days of their first insemination. The 25-day interval from breeding topregnancy detection is shorter than current methods and the 30-dayinterval from first breeding to second breeding for nonpregnant cows ismuch shorter than the industry average.

Pregnancy testing in dairy cows has usually been done by manuallyfeeling for an embryo in the uterus. The manual test is typically done35 to 60 days after breeding. On large dairies, a single veterinarianmay be employed 100% time to do manual pregnancy testing. The onlyalternative to manual testing is ultrasound testing. This can be done at28 days after breeding. Ultrasound testing is not routine because theequipment is expensive and the test takes longer than the manual test.

F. Drugs used to manipulate reproductive cycles in dairy

Dairy cows can be injected with prostaglandin F_(2α) (PGF) to regressthe corpus luteum and cause estrus. PGF only works if the cow has acorpus luteum. Cows that do not have a corpus luteum will not respond toPGF. Dairy cows without a corpus luteum can be injected withgonadotropin-releasing hormone (GnRH) to cause ovulation and theformation of a corpus luteum. One typical way to manage dairy cowswithout a corpus luteum is to inject GnRH, wait 7 days (allows CL toform), inject PGF and await the cow's next estrus. Both PGF and GnRH areinexpensive and are commonly used in dairy herds (either alone or incombination). Another approach is to inject GnRH, wait seven days andinject PGF, and then wait two days and inject GnRH. This protocol(Ovsynch protocol) is popular because cows can be inseminated after thesecond GNRH without the need for estrus detection.

G. Implementation of Improved Pregnancy Tests in Breeding Programs

Using the new assays, there are four possible outcomes with respect tothe PAG and progesterone results: +/+, ±, ∓ and −/−. Based on theresults, various steps will be desired for implementation of breedingprograms. The different possibilities and the likely desired course ofaction are set forth below in Table 1. TABLE 1 Reproductive planimplemented 25 days after breeding PAG Test Progesterone PregnancyResult Test Result outcome Farmer action Positive Positive Cow is Noaction needed. Farmer pregnant is happy. Positive Negative The embryoCow does not have a CL underwent (based on low progesterone). earlyInject GnRH (cause ovulation), embryonic wait seven days, inject PGFdeath and (regress CL). The farmer can the cow breed at estrus or analterna- is not tive would be to give another pregnant. injection ofGnRH at 48 hours after PGF to induce ovulation and breed. These arecommon reproductive management treatments in dairy. Negative PositiveCow is not Cow has a CL but does not pregnant have an embryo. Inject PGFto regress the CL. The farmer can breed at estrus or an alterna- tivewould be give another injection of GnRH at 48 hours after PGF to induceovulation and breed. These are common reproductive management treatmentsin dairy Negative Negative Cow is not Cow does not have a CL andpregnant does not have an embryo. Inject GnRH, wait seven days, injectPGF. The farmer can breed at estrus or an alterna- tive would be giveanother injection of GnRH at 48 hours after PGF to induce ovulation andbreed. These are common reproductive management treatments in dairy.

II. PREGNANCY ASSOCIATED GLYCOPROTEINS

The placenta is the hallmark of the eutherian mammal. Rather than beingthe most anatomically conserved mammalian organ, however, it arguably isthe most diverse (Haig, 1993). Placentation ranges from the invasivehemochorial type, as in the human, where the trophoblast surface is indirect contact with maternal blood, to the epitheliochorial (e.g., pig),where the uterine epithelium is not eroded (Amoroso, 1952). Not only isplacental structure highly variable, the polypeptide hormones theplacenta produces also vary between species (Haig, 1993; Roberts et al.,1996). For example, no group of mammals other than higher primatespossesses a chorionic gonadotrophin homologous to hCG for luteal supportin early pregnancy, and only the ruminant ungulates are known to produceType I interferon as an antilyteolytic hormone (Roberts et al., 1996).

Placentation in ruminants, such as cattle and sheep, is superficial,relatively noninvasive, and known as synepitheliochorial cotyledonary(Wooding, 1992). ‘Synepitheliochorial’ describes the fetal-maternalsyncytium formed by the fusion of trophoblast binucleate cells anduterine epithelial cells, whereas, ‘cotyledonary’ describes the grossstructure of the placenta and specifically the tufts of villoustrophoblast (cotyledons) that insinuate themselves into the crypts ofthe maternal caruncles. These regions of interdigitated and partiallyfused fetal cotyledonary and maternal caruncles are the placentomes andare the main sites for nutrient and gas exchange in the placenta. Thebinucleate cells, which compose about 20% of the surface epithelium(trophectoderm) migrate and fuse with maternal uterine epithelial cellsand deliver their secretory products directly to the maternal system.Among the products are the placental lactogens (Wooding, 1981) and thepregnancy-associated glycoproteins (Zoli et al., 1992a.) Bovinepregnancy-associated glycoproteins (boPAGs), also known under a varietyof other names including pregnancy-specific protein-B (Butler et al.,1982), were discovered in attempts to develop pregnancy tests forlivestock (Sasser et al., 1986; Zoli et al., 1991; Zoli et al., 1992a).Rabbits were injected with extracts of placental cotyledons, andantibodies not directed against placental antigens were removed byadsorption with tissue extracts from nonpregnant animals. The resultingantisera provided the basis of an accurate pregnancy test for cattle andsheep as early as one month post-insemination.

Xie et al. (1991) used an antiserum directed against purified boPAGsfrom cattle and from sheep to screen cDNA libraries from late placentaltissue. The fill-length cDNAs shared 86% nucleotide sequence identitieswith each other and a surprising 60% sequence identity to pepsinogens.The boPAGs had mutations in and around their active sites that wouldrender them inactive as proteinases (Xie et al., 1991; Guruprasad etal., 1996). The similarities to pepsin A (˜50% amino acid identity) andchymosin (˜45%) in primary structure has allowed atomic models of ovine(ov)PAG1 and boPAG1 to be built (Guruprasad et al., 1996). Bothmolecules have the bilobed structure typical of all known eukaryoticaspartic proteinases and possess a cleft between the two lobes capableof accommodating peptides up to 7 amino acids long. Modeling stronglysuggested that both ovPAGl and boPAG1 can bind the pepsin inhibitorpepstatin, a prediction that has been validated.

Even in initial studies (Butler et al., 1982; Zoli et al., 1991; Xie etal., 1991; Xie et al., 1994; Xie et al., 1996), it was clear that theboPAGs were heterogenous in molecular weight and charge, and as moreisoforms have been purified it has become evident that they differ intheir amino terminal sequences (Atkinson et al., 1993; Xie et al.,1997a). Further library screening has revealed additional transcripts inruminants (Xie et al., 1994; Xie et al., 1995; Xie et al., 1997b) andthe existence of PAGs in non-ruminant species such as the pig(Szafranska et al., 1995), and the horse (Guruprasad et al., 1996).

Despite their apparent lack of proteolytic activity, all of the PAGswhose amino terminal sequences have been determined are proteolyticallyprocessed in a manner typical of other aspartic proteases such as pepsin(Davies, 1990). For example, a pro-peptide of most PAGs, whichconstitutes the first 38 amino acids of the secreted form and whichnormally folds into the active site region, has been cleaved from thesecreted forms of PAG. Thus, the calculated molecular weight of themature, non-glycosylated PAG, i.e. with signal sequence propeptideremoved would be 36,000 daltons and the circulating antigen in serumwould also lack this segment. The observed molecular weight of secretedPAG, however, is much larger ranging from 45,000 daltons to 90,000daltons (Xie et al., 1991; Sasser et al., 1989; Xie et al., 1996),probably due to extensive glycosylation (Holdsworth et al., 1982).Multiple boPAG genes in the bovine genome have most likely contributedto the triphasic alterations of PAG concentrations in maternal serum.

A. BoPAG1

Bovine (bo) PAG1 was initially identified as a unique placental antigenby raising antisera to total bovine placental extracts (Zoli et al.,1991). It is a product of binucleate trophoblast cells (Xie et al.,1991; Zoli et al., 1992b) which constitute the invasive component of theplacenta (Wooding, 1992; Guillomot, 1995). In 1991, cDNA for both boPAG1and ovine PAG1 was identified (ovPAG1) (Xie et al., 1991). Surprisingly,the PAG1 belong to the aspartic proteinase (AP) gene family, a groupingthat includes pepsin, chymosin, renin, and cathepsin D and E (Guruprasadet al., 1996). Unlike other members of the AP family, both ovPAG1 andboPAG1 appear to be enzymatically inactive, since the catalytic domainin the active site region is mutated (Xie et al., 1991; Guruprasad etal., 1996).

BoPAG1 gene contains 9 exons and 8 introns (Xie et al., 1996), anidentical organization to that of other mammalian aspartic genes.Southern genomic blotting with a probe encompassing exon 7 and exon 8,which represent the most conserved region of PAG relative to other AP,indicated that there were probably many PAG genes. In addition, when abovine genomic library was probed with boPAGl cDNA, 0.06% positive phageplaques were identified, suggesting that there may be 100 or more PAGgenes in the bovine genome (Xie et al., 1995). This approximation hasrecently been confumed by a variety of other approaches (Xie et al.,1997b).

Levels of boPAG1 or related molecules that cross-react with a boPAG-1antiserum are very low around day 21 to day 27 (Warnick et al., 1995;Beal et al., 1992; Cameron and Malmo, 1993; Butler et al., 1982), aremaintained at a higher, but still low concentration until about day 100of the pregnancy and then rise quickly to ˜100 ng/ml. The concentrationsthen remain relatively constant until the last quarter of pregnancy whenthey peak at 1 μg/ml of serum or greater right before parturition. Oneexplanation for the triphasic profile of boPAG1 immunoreactivity is thatexpression of boPAG1 is very low in early pregnancy, rises considerablyat mid gestation and peaks before parturition (Sasser et al., 1986; Zoliet al., 1992a; Patel et al., 1995). Alternatively, the presence ofimmunoreactive antigen in very early pregnancy may be due to theproduction of other boPAGs. The rise in the second trimester may reflectproduction of yet a different class of boPAG or possibly the initiationof low PAG1 expression. The exponential rise of boPAGs just prior toparturition could represent a sudden increase in the synthesis of one ormore boPAG1 related molecules or increased “escape” across a leakierutero-placental junction.

Immunocytochemistry and in situ hybridization analyses have shown thatboPAG1 and ovPAG1, and their close relatives (since neither the antiseranor the probes are expected to be monospecific) are localized tobinucleate cells (Xie et al., 1991; Zoli et al., 1992b) In contrast, theantigenically distinct boPAG2 is expressed in predominantly mononucleatecells of the trophectoderm (Xie et al., 1994). In the ruminants,binucleate cells are the invasive components of the trophoblast and donot appear until about day 13 in sheep and day 17 in cattle (Wooding,1992). They then quickly increase in number. By day 21 in cattle theyconstitute up to 20% of cells in the trophectoderm, and a highpercentage are actively fusing with maternal uterine epithelial cells(Wooding, 1992; King et al., 1980; Guillomot, 1995). Binucleate cellgranules, which contain PAG1 (Zoli et al., 1992b), are discharged fromthe fusion cell towards the matemal stroma and its network ofcapillaries. Therefore, the binucleate cell products have ready accessto the maternal circulation.

B. Novel OvPAG and BoPAG Species

cDNA for a series of novel boPAGs have been identified and cloned. Asimilar large family of ovine (ov) PAGs have been identified from sheepplacenta (Xie et al., 1991; Xie et al., 1997a; Xie et al., 1997b).Certain of the boPAGs are useful in detection of early pregnancy incattle. These molecules are homologous to, but different from, boPAG1(Xie et al., 1991). Apparently, PAGs constitute a polymorphic group (Xieet al., 1994; Xie et al., 1995; Xie et al., 1997a; Xie et al., 1997b),whose members either show variable degrees of immunocrossreactivity ordo not cross-react at all with the antisera that have been developed.Some of the cloned PAGs are only expressed in binucleate cells of theplacental. These cells are known to have an endocrine function (Wooding,1992). They produce placental lactogen and steroids, for example.However, the fimctions of the PAG family members are unknown, althoughthey enter the maternal circulation.

One important aspect of the present invention is that PAGs are notexpressed uniformly throughout pregnancy. Some are found early inpregnancy, while are others are expressed in later stages. For example,PAGs that are expressed most strongly in the invasive binucleate cellsat implantation are not dominant in late pregnancy. Conversely, boPAG1(PSP-B) (Xie et al., 1991; Butler et al., 1982; Sasser et al., 1986)primarily is a product of binucleate cells of the late placenta, andantiserum raised against it fails to recognize the dominant PAG producedby binucleate cells in early pregnancy. Therefore, the test developed bythe other groups and based on boPAG1/PSP-B/PSP60 (Butler et al., 1982;Sasser et al., 1986; Zoli et al., 1992a; Mialon et al., 1993; Kiracofeet al., 1994 ) is only marginally useful early in pregnancy because theantigen is produced in extremely small amounts, if at all, at that time.The expression pattern of boPAG1 also helps explain the concentrationprofile of the antigen measured in serum. At term, levels can exceed 5μg/ml, while at day 40, when the development of the placenta in terms ofsize is almost complete, concentrations are around 10 ng/ml, i.e.,500-fold lower.

Certain of the novel boPAGs disclosed in this invention (boPAG 4, 5, 6,7, and 9), having the sequences of SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, and SEQ ID NO:32 are present at day 25 ofpregnancy. These PAGs are expressed in invasive binucleate cells whichrelease their secretory granules into maternal uterine capillary bed. Ofthese five, boPAG4 appears to cross react with the late pregnancy PAG,boPAG1, which has been the basis of the earlier pregnancy test. Byvirtue of their early expression, these PAGs can be detected byconventional immunological techniques in physiological fluids of heifersor cows (especially in serum, urine, and milk) to detect the presence ofa fetus or fetuses in the uterus prior to day 30 of pregnancy. Thus, thepresence of these antigens provide a diagnostic test of early pregnancyin cattle.

Similar observations on the diversity of PAGs, the localization ofdifferent PAGs to either mononucleated and binucleated cells, and thelikely varied timing of PAG expression have been noted in sheep (Xie etal., 1991; Xie et al., 1997a; Xie et al., 1997b). Because of the largenumber of genes noted in other species, these observations are likelyalso to hold for other Artiodactyla, as well.

C. Purification of the Proteins

It will be desirable to purify the various PAGs. Protein purificationtechniques are well known to those of skill in the art. These techniquesinvolve, at one level, the crude fractionation of the cellular milieu topolypeptide and non-polypeptide fractions. Having separated thepolypeptide from other proteins, the polypeptide of interest may befurther purified using chromatographic and electrophoretic techniques toachieve partial or complete purification (or purification tohomogeneity). Analytical methods particularly suited to the preparationof a pure peptide are ion-exchange chromatography, exclusionchromatography; polyacrylamide gel electrophoresis; isoelectricfocusing. A particularly efficient method of purifying peptides is fastprotein liquid chromatography or even HPLC.

Certain aspects of the present invention concern the purification, andin particular embodiments, the substantial purification, of an encodedprotein or peptide. The term “purified protein or peptide” as usedherein, is intended to refer to a composition, isolatable from othercomponents, wherein the protein or peptide is purified to any degreerelative to its naturally-obtainable state. A purified protein orpeptide therefore also refers to a protein or peptide, free from theenvironment in which it may naturally occur.

Generally, “purified” will refer to a protein or peptide compositionthat has been subjected to fractionation to remove various othercomponents, and which composition substantially retains its expressedbiological activity. Where the term “substantially purified” is used,this designation will refer to a composition in which the protein orpeptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95% or more of the proteins in the composition.

Various methods for quantifying the degree of purification of theprotein or peptide will be known to those of skill in the art in lightof the present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. A preferred methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity, hereinassessed by a “-fold purification number” (i.e., 2-fold, 5-fold,10-fold, 50-fold, 100-fold, 1000-fold, etc.). The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification and whetheror not the expressed protein or peptide exhibits a detectable activity.

Various techniques suitable for use in protein purification will be wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies and the like or byheat or acid pH denaturation of contaminating proteins, followed bycentrifugation; chromatography steps such as ion exchange, gelfiltration, reverse phase, hydroxylapatite and affinity chromatography;isoelectric focusing; gel electrophoresis; and combinations of such andother techniques. As is generally known in the art, it is believed thatthe order of conducting the various purification steps may be changed,or that certain steps may be omitted, and still result in a suitablemethod for the preparation of a substantially purified protein orpeptide.

There is no general requirement that the protein or peptide always beprovided in their most purified state. Indeed, it is contemplated thatless substantially purified products will have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater “-fold” purification thanthe same technique utilizing a low pressure chromatography system.Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein.

It is known that the migration of a polypeptide can vary, sometimessignificantly, with different conditions of SDS/PAGE and according tohow extensively it is glycosylated (Capaldi et al., 1977). It willtherefore be appreciated that under differing electrophoresisconditions, the apparent molecular weights of purified or partiallypurified expression products may vary.

High Performance Liquid Chromatography (HPLC) is characterized by a veryrapid separation with extraordinary resolution of peaks. This isachieved by the use of very fine particles and high pressure to maintainan adequate flow rate. Separation can be accomplished in a matter ofmin, or at most an hour. Moreover, only a very small volume of thesample is needed because the particles are so small and close-packedthat the void volume is a very small fraction of the bed volume. Also,the concentration of the sample need not be very great because the bandsare so narrow that there is very little dilution of the sample.

Gel chromatography, or molecular sieve chromatography, is a special typeof partition chromatography that is based on molecular size. The theorybehind gel chromatography is that the column, which is prepared withtiny particles of an inert substance that contain small pores, separateslarger molecules from smaller molecules as they pass through or aroundthe pores, depending on their size. As long as the material of which theparticles are made does not adsorb the molecules, the sole factordetermining rate of flow is the size. Hence, molecules are eluted fromthe column in decreasing size, so long as the shape is relativelyconstant. Gel chromatography is unsurpassed for separating molecules ofdifferent size because separation is independent of all other factorssuch as pH, ionic strength, temperature, etc. There also is virtually noadsorption, less zone spreading and the elution volume is related tomolecular weight.

Afinity Chromatography is a chromatographic procedure that relies on thespecific affinity between a substance to be isolated and a molecule thatit can specifically bind to. This is a receptor-ligand type interaction.The column material is synthesized by covalently coupling one of thebinding partners to an insoluble matrix. The column material is thenable to specifically adsorb the substance from the solution. Elutionoccurs by changing the conditions to those in which binding will notoccur (alter pH, ionic strength, temperature, etc.).

A particular type of affinity chromatography useful in the purificationof carbohydrate containing compounds is lectin affinity chromatography.Lectins are a class of substances that bind to a variety ofpolysaccharides and glycoproteins. Lectins are usually coupled toagarose by cyanogen bromide. Conconavalin A coupled to Sepharose was thefirst material of this sort to be used and has been widely used in theisolation of polysaccharides and glycoproteins other lectins that havebeen include lentil lectin, wheat germ agglutinin which has been usefulin the purification of N-acetyl glucosaminyl residues and Helix pomatialectin. Lectins themselves are purified using affinity chromatographywith carbohydrate ligands. Lactose has been used to purify lectins fromcastor bean and peanuts; maltose has been useful in extracting lectinsfrom lentils and jack bean; N-acetyl-D galactosamine is used forpurifying lectins from soybean; N-acetyl glucosaminyl binds to lectinsfrom wheat gern; D-galactosamine has been used in obtaining lectins fromclams and L-fucose will bind to lectins from lotus. PAG antigens can bepurified by using a pepstatin agarose affinity matrix, e.g., asdescribed by Avalle et al. (2001)

The matrix should be a substance that itself does not adsorb moleculesto any significant extent and that has a broad range of chemical,physical and thermal stability. The ligand should be coupled in such away as to not affect its binding properties. The ligand should alsoprovide relatively tight binding. And it should be possible to elute thesubstance without destroying the sample or the ligand. One of the mostcommon forms of affinity chromatography is immunoaffinitychromatography. The generation of antibodies that would be suitable foruse in accord with the present invention is discussed below.

D. Antigen Compositions

The present invention provides for the use of PAGs or peptides asantigens for the generation of polyclonal antisera and monoclonalantibodies for use in the detection of pregnancy. It is envisioned thatsome variant of a PAG, or portions thereof, will be coupled, bonded,bound, conjugated or chemically-linked to one or more agents vialinkers, polylinkers or derivatized amino acids. This may be performedsuch that a bispecific or multivalent composition or vaccine isproduced. It is further envisioned that the methods used in thepreparation of these compositions will be familiar to those of skill inthe art and should be suitable for administration to animals, i.e.,pharmaceutically acceptable. Preferred agents are the carriers such askeyhole limpet hemocyannin (KLH) or glutathione-S-transferase.

In order to formulate PAGs for immunization, one will generally desireto employ appropriate salts and buffers to render the polypeptidesstable. Aqueous compositions of the present invention comprise aneffective amount of the PAG antigen to the host animal, dissolved ordispersed in a pharmaceutically acceptable carrier or aqueous medium.Such compositions may be referred to as inocula. The phrase“pharmaceutically or pharmacologically acceptable” refer to molecularentities and compositions that do not produce adverse, allergic, orother untoward reactions when administered to an animal or a human. Asused herein, “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknow in the art. Except insofar as any conventional media or agent isincompatible with the vectors or cells of the present invention, its usein therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

The compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention will be via any common route so longas the target tissue is available via that route. This includes oral,nasal, buccal, rectal, vaginal or topical. Alternatively, administrationmay be by orthotopic, intradermal, subcutaneous, intramuscular,intraperitoneal or intravenous injection. Such compositions wouldnormally be administered as pharmaceutically acceptable compositions,described supra.

The PAGs also may be administered parenterally or intraperitoneally.Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It should be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial an antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the PAGs inthe required amount in the appropriate solvent with various of the otheringredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle whichcontains the basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum-drying and freeze-drying techniques which yield apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

The compositions of the present invention may be formulated in a neutralor salt form. Phannaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, preparations should meet applicable sterility, pyrogenicity,general safety and purity standards.

III. GENERATING ANTIBODIES REACTIVE WITH PAGS AND PROGESTERONE

In another aspect, the present invention contemplates an antibody thatis immunoreactive with a PAG molecule or progesterone of the presentinvention, or any portion thereof. An antibody can be a polyclonal or amonoclonal antibody composition, both of which are preferred embodimentsof the present invention. Means for preparing and characterizingantibodies are well known in the art (see, e.g., Harlow and Lane, 1988).

Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogen comprising a peptide or polypeptide of the presentinvention and collecting antisera from that immunized animal. A widerange of animal species can be used for the production of antisera.Typically an animal used for production of anti-antisera is a non-humananimal including rabbits, mice, rats, hamsters, pigs or horses. Becauseof the relatively large blood volume of rabbits, a rabbit is a preferredchoice for production of polyclonal antibodies.

Antibodies, both polyclonal and monoclonal, specific for isoforms ofantigen may be prepared using conventional immunization techniques, aswill be generally known to those of skill in the art. A compositioncontaining antigenic epitopes of the compounds of the present inventioncan be used to immunize one or more experimental animals, such as arabbit or mouse, which will then proceed to produce specific antibodiesagainst the compounds of the present invention. Polyclonal antisera maybe obtained, after allowing time for antibody generation, simply bybleeding the animal and preparing serum samples from the whole blood.

It is proposed that the monoclonal antibodies of the present inventionwill find useful application in standard immunochemical procedures, suchas ELISA and Western blot methods and in immunohistochemical proceduressuch as tissue staining, as well as in other procedures which mayutilize antibodies specific to PAG-related antigen epitopes.Additionally, it is proposed that monoclonal antibodies specific to theparticular PAG of different species may be utilized in other usefulapplications.

In general, both polyclonal and monoclonal antibodies against PAG orprogesterone may be used in a variety of embodiments. For example, theymay be employed in antibody cloning. protocols to obtain cDNAs or genesencoding antibodies to PAG(s) and progesterone. They may also be used ininhibition studies to analyze the effects of PAG or progesterone relatedpeptides in cells or animals. Anti-PAG or antibodies to progesteronepathway enzymes will also be usefull in immunolocalization studies toanalyze the distribution of PAGs or enzymes that participate inprogesterone biosynthesis or metabolism during various cellular events,for example, to determine the cellular or tissue-specific distributionof PAGs or progesterone biosynthesis or metabolism under differentpoints in the cell cycle. A particularly useful application of suchantibodies is in purifying native or recombinant PAG, for example, usingan antibody affinity column. The operation of all such immunologicaltechniques will be known to those of skill in the art in light of thepresent disclosure.

Means for preparing and characterizing antibodies are well known in theart (see, e.g., Harlow and Lane, 1988; incorporated herein byreference). More specific examples of monoclonal antibody preparationare give in the examples below.

As is well known in the art, a given composition may vary in itsimmunogenicity. It is often necessary therefore to boost the host immunesystem, as may be achieved by coupling a peptide or polypeptideimmunogen to a carrier. Exemplary and preferred carriers are keyholelimpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albuminssuch as ovalbumin, mouse serum albumin or rabbit serum albumin can alsobe used as carriers. Means for conjugating a polypeptide to a carrierprotein are well known in the art and include glutaraldehyde,m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide andbis-biazotized benzidine.

As also is well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Exemplary andpreferred adjuvants include complete Freund's adjuvant (a non-specificstimulator of the immune response containing killed Mycobacteriumtuberculosis), incomplete Freund's adjuvants and aluminum hydroxideadjuvant.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen (subcutaneous, intramuscular, intradermal, intravenous andintraperitoneal). The production of polyclonal antibodies may bemonitored by sampling blood of the immunized animal at various pointsfollowing immunization. A second, booster, injection may also be given.The process of boosting and titering is repeated until a suitable titeris achieved. When a desired level of immunogenicity is obtained, theimmunized animal can be bled and the serum isolated and stored, and/orthe animal can be used to generate mAbs.

MAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g. a purified orpartially purified PAG or progesterone. The immunizing composition isadministered in a manner effective to stimulate antibody producingcells. Rodents such as mice and rats are preferred animals, however, theuse of rabbit, sheep or frog cells is also possible. The use of rats mayprovide certain advantages (Goding, 1986), but mice are preferred, withthe BALB/c mouse being most preferred as this is most routinely used andgenerally gives a higher percentage of stable fusions.

Following immunization, somatic cells with the potential for producingantibodies, specifically B-lymphocytes (B-cells), are selected for usein the mAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible. Often, a panel of animals will have beenimmunized and the spleen of animal with the highest antibody titer willbe removed and the spleen lymphocytes obtained by homogenizing thespleen with a syringe. Typically, a spleen from an immunized mousecontains approximately 5×10⁷ to 2×10⁸ lymphocytes.

The antibody-producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render then incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, 1986; Campbell, 1984). For example, wherethe immunized animal is a mouse, one may use P3-X63/Ag8, P3-X63-Ag8.653,NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 andS194/5XXO Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all usefulin connection with cell fusions.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 ratio, though the ratio may vary from about 20:1to about 1:1, respectively, in the presence of an agent or agents(chemical or electrical) that promote the fusion of cell membranes.Fusion methods using Sendai virus have been described (Kohler andMilstein, 1975; 1976), and those using polyethylene glycol (PEG), suchas 37% (v/v) PEG, by Gefter et al., (1977). The use of electricallyinduced fusion methods is also appropriate (Goding, 1986).

Fusion procedures usually produce viable hybrids at low frequencies,around 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfulsedcells particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de izovo synthesis of both purinesand pyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B-cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B-cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide mAbs. The cell lines may be exploitedfor mAb production in two basic ways. A sample of the hybridoma can beinjected (often into the peritoneal cavity) into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide mAbs in high concentration. The individualcell lines could also be cultured in vitro, where the mAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. mAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.

IV. ASSAYS FOR PAG AND PROGESTERONE EXPRESSION FOR THE DETECTION OFPREGNANCY

According to the present invention, the present inventors havedetermined that PAGs in combination with progesterone can be usedadvantageously expressed in early stages of pregnancy and, therefore,can be used as markers in the detection of pregnancy at an early stage.In cattle, the BoPAGs may be used individually or in combination toprovide a diagnostic evaluation of pregnancy. According to the presentinvention, these boPAGs include BoPAGs1 through 21. Other boPAGs, andPAGs from other species, may prove useful, alone or in combination, forsimilar purposes.

A. Immunologic Detection of BoPAGs and Progesterone

The present invention entails the use of antibodies in the immunologicdetection of PAGs or progesterone. Various useful immunodetectionmethods have been described in the scientific literature, such as, e.g.,Nakamura et al. (1987; incorporated herein by reference). Immunoassays,in their most simple and direct sense, are binding assays. Certainpreferred immunoassays are the various types of enzyme linkedimmunosorbent assays (ELISAs) and radioimmunoassays (RIA).Immunohistochemical detection using tissue sections also is particularlyuseful. However, it will be readily appreciated that detection is notlimited to such techniques, and Western blotting, dot blotting, FACSanalyses, and the like also may be used in connection with the presentinvention.

In general, immunobinding methods include obtaining a sample suspectedof containing a protein, peptide or antibody, and contacting the samplewith an antibody or protein or peptide in accordance with the presentinvention, as the case may be, under conditions effective to allow theformation of immunocomplexes. Preferred samples, according to thepresent invention, are fluids, such as milk, urine, blood, serum orsaliva.

Contacting the chosen biological sample with the protein, peptide orantibody under conditions effective and for a period of time sufficientto allow the formation of immune complexes (primary immune complexes) isgenerally a matter of simply adding the composition. to the sample andincubating the mixture for a period of time long enough for theantibodies to form immune complexes with PAGs or progesterone. Afterthis time, the PAG- or progesterone antibody mixture will be washed toremove any non-specifically bound antibody species, allowing only thoseantibodies specifically bound within the primary immune complexes to bedetected.

In general, the detection of immunocomplex formation is well known inthe art and may be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any radioactive, fluorescent, biological orenzymatic tags or labels of standard use in the art. U.S. Patentsconcerning the use of such labels include U.S. Pat. Nos. 3,817,837;3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241,each incorporated herein by reference. Of course, one may findadditional advantages through the use of a secondary binding ligand suchas a second antibody or a biotin/avidin ligand binding arrangement, asis known in the art.

Usually, the primary immune complexes may be detected by means of asecond binding ligand that has binding affinity for the PAG- orprogesterone-specific first antibody. In these cases, the second bindingligand may be linked to a detectable label. The second binding ligand isitself often an antibody, which may thus be termed a “secondary”antibody. The primary immune complexes are contacted with the labeled,secondary binding ligand, or antibody, under conditions effective andfor a period of time sufficient to allow the formation of secondaryimmune complexes. The secondary immune complexes are then generallywashed to remove any non-specifically bound labeled secondary antibodiesor ligands, and the remaining label in the secondary immune complexes isthen detected.

Further methods include the detection of primary immune complexes by atwo step approach. A second binding ligand, such as an antibody, thathas binding affinity for the PAG or progesterone antibody is used toform secondary immune complexes, as described above. The second bindingligand contains an enzyme capable of processing a substrate to adetectable product and, hence, amplifying signal over time. Afterwashing, the secondary immune complexes are contacted with substrate,permitting detection.

Progesterone can also be detected in accordance with the invention usingvarious commercially available detection kits. For example, theCoat-a-Count™ progesterone kit used by the inventors, which is availablefrom Diagnostics Products Corporation (Los Angeles, Calif.). Examples ofother assays that have been described include the immunoenzymatictechnique described, for example, by Stefanakis et al., (1994) and byStanley et al. (1986); and salivary progesterone level assays desribed,for example, by Lu et al., (1997) and Vienravi et al., 1994.

B. ELISA

As a part of the practice of the present invention, the principles of anenzyme-linked immunoassay (ELISA) may be used. ELISA was firstintroduced by Engvall and Perlmann (1971) and has become a powerfulanalytical tool using a variety of protocols (Engvall, 1980; Engvall,1976; Engvall, 1977; Gripenberg et al., 1978; Sarngadharan et al.,1984). ELISA allows for substances to be passively adsorbed to solidsupports such as plastic to enable facile handling under laboratoryconditions. For a comprehensive treatise on ELISA the skilled artisan isreferred to “ELISA; Theory and Practise” (Crowther, 1995 incorporatedherein by reference).

The sensitivity of ELISA methods is dependent on the turnover of theenzyme used and the ease of detection of the product of the enzymereaction. Enhancement of the sensitivity of these assay systems can beachieved by the use of fluorescent and radioactive substrates for theenzymes. Immunoassays encompassed by the present invention include, butare not limited to those described in U.S. Pat. No. 4,367,110 (doublemonoclonal antibody sandwich assay) and U.S. Pat. No. 4,452,901 (westernblot). Other assays include immunoprecipitation of labeled ligands andimmunocytochemistry, both in vitro and in vivo.

In a preferred embodiment, the invention comprises a “sandwich” ELISA,where anti-PAG antibodies are immobilized onto a selected surface, suchas a well in a polystyrene microtiter plate or a dipstick. Then, a testcomposition suspected of containing PAGs, e.g., a clinical sample, iscontacted with the surface. After binding and washing to removenon-specifically bound immunocomplexes, the bound antigen may bedetected by a second antibody to the PAG.

In another exemplary ELISA, polypeptides from the sample are immobilizedonto a surface and then contacted with the anti-PAG antibodies. Afterbinding and washing to remove non-specifically bound immune complexes,the bound antibody is detected. Where the initial antibodies are linkedto a detectable label, the primary immune complexes may be detecteddirectly. Alternatively, the immune complexes may be detected using asecond antibody that has binding affinity for the first antibody, withthe second antibody being linked to a detectable label.

Another ELISA in which the PAGs are immobilized involves the use ofantibody competition in the detection. In this ELISA, labeled antibodiesare added to the wells, allowed to bind to the PAG, and detected bymeans of their label. The amount of PAG in a sample is determined bymixing the sample with the labeled antibodies before or duringincubation with coated wells. The presence of PAG in the sample acts toreduce the amount of antibody available for binding to the well, andthus reduces the ultimate signal.

Irrespective of the format employed, ELISAs have certain features incommon, such as coating, incubating or binding, washing to removenon-specifically bound species, and detecting the bound immunecomplexes. In coating a plate with either antigen or antibody, one willgenerally incubate the wells of the plate with a solution of the antigenor antibody, either overnight or for a specified period of hours. Thewells of the plate will then be washed to remove incompletely adsorbedmaterial. Any remaining available surfaces of the wells are then“coated” with a nonspecific protein that is antigenically neutral withregard to the test antisera. These include bovine serum albumin (BSA),casein and solutions of milk powder. The coating allows for blocking ofnonspecific adsorption sites on the immobilizing surface and thusreduces the background caused by nonspecific binding of antisera ontothe surface.

In ELISAs, it is probably more customary to use a secondary or tertiarydetection means rather than a direct procedure. Thus, after binding of aprotein or antibody to the well, coating with a non-reactive material toreduce background, and washing to remove unbound material, theimmobilizing surface is contacted with the control human cancer and/orclinical or biological sample to be tested under conditions effective toallow immune complex (antigen/antibody) formation. Detection of theimmune complex then requires a labeled secondary binding ligand orantibody, or a secondary binding ligand or antibody in conjunction witha labeled tertiary antibody or third binding ligand.

“Under conditions effective to allow immune complex (antigen/antibody)formation” means that the conditions preferably include diluting theantigens and antibodies with solutions such as BSA, bovine gammaglobulin (BGG), evaporated or powdered milk, and phosphate bufferedsaline (PBS)/Tween. These added agents also tend to assist in thereduction of nonspecific background.

The “suitable” conditions also mean that the incubation is at atemperature and for a period of time sufficient to allow effectivebinding. Incubation steps are typically from about 1 h to 2 h to 4 h, attemperatures preferably on the order of 25° C. to 27° C., or may beovernight at about 4° C. or so.

To provide a detecting means, the second or third antibody will have anassociated label to allow detection. Preferably, this will be an enzymethat will generate color development upon incubating with an appropriatechromogenic substrate. Thus, for example, one will desire to contact andincubate the first or second immune complex with a urease, glucoseoxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibodyfor a period of time and under conditions that favor the development offurther immunecomplex formation (e.g. incubation for 2 h at roomtemperature in a PBS-containing solution such as PBS-Tween).

After incubation with the labeled antibody, and subsequent to washing toremove unbound material, the amount of label is quantified, e.g., byincubation with a chromogenic substrate such as urea and bromocresolpurple or 2,2′-azido-di-(3-ethyl-benzthiazoline-6-sulfonic acid [ABTS]and H₂O₂, in the case of peroxidase as the enzyme label. Quantitation isthen achieved by measuring the degree of color generation, e.g., using avisible spectra spectrophotometer.

A variant of ELISA is the enzyme-linked coagulation assay, or ELCA (U.S.Pat. No. 4,668,621), which uses the coagulation cascade combined withthe labeling enzyme RW-XA as a universal detection system. The advantageof this system for the current invention, is that the coagulationreactions can be performed at physiological pH in the presence of a widevariety of buffers. It is therefore possible to retain the integrity ofcomplex analytes.

C. Nucleic Acid Detection

In a variety of embodiments, it will be desirable to detect nucleicacids (mRNAs or cDNAs) for BoPAGs and/or progesterone and determine thelevels of the corresponding proteins. Such methods include Northernassays and RT-PCR. The following describe methods relevant to thedetection and quantification of such nucleic acids.

1. Hybridization

The use of a probe or primer of between 13 and 100 nucleotides,preferably between 17 and 100 nucleotides in length, or in some aspectsof the invention up to 1-2 kilobases or more in length, allows theformation of a duplex molecule that is both stable and selective.Molecules having complementary sequences over contiguous stretchesgreater than 20 bases in length are generally preferred, to increasestability and/or selectivity of the hybrid molecules obtained. One willgenerally prefer to design nucleic acid molecules for hybridizationhaving one or more. complementary sequences of 20 to 30 nucleotides, oreven longer where desired. Such fragments may be readily prepared, forexample, by directly synthesizing the fragment by chemical means or byintroducing selected sequences into recombinant vectors for recombinantproduction.

Accordingly, the nucleotide sequences of the invention may be used fortheir ability to selectively form duplex molecules with complementarystretches of DNAs and/or RNAs or to provide primers for amplification ofDNA or RNA from samples. Depending on the application envisioned, onewould desire to employ varying conditions of hybridization to achievevarying degrees of selectivity of the probe or primers for the targetsequence.

For applications requiring high selectivity, one will typically desireto employ relatively high stringency conditions to form the hybrids. Forexample, relatively low salt and/or high temperature conditions, such asprovided by about 0.02 M to about 0.10 M NaCl at temperatures of about50° C. to about 70° C. Such high stringency conditions tolerate little,if any, mismatch between the probe or primers and the template or targetstrand and would be particularly suitable for isolating specific genesor for detecting specific MRNA transcripts. It is generally appreciatedthat conditions can be rendered more stringent by the addition ofincreasing amounts of formamide.

Conditions may be rendered less stringent by increasing saltconcentration and/or decreasing temperature. For example, a mediumstringency condition could be provided by about 0.1 to 0.25 M NaCl attemperatures of about 37° C. to about 55° C., while a low stringencycondition could be provided by about 0.15 M to about 0.9 M salt, attemperatures ranging from about 20° C. to about 55° C. Hybridizationconditions canbe readily manipulated depending on the desired results.

In other embodiments, hybridization may be achieved under conditions of,for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 1.0 mMdithiothreitol, at temperatures between approximately 20° C. to about37° C. Other hybridization conditions utilized could includeapproximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 MM MgCl₂, attemperatures ranging from approximately 40° C. to about 72° C.

In certain embodiments, it will be advantageous to employ nucleic acidsof defined sequences of the present invention in combination with anappropriate means, such as a label, for determining hybridization. Awide variety of appropriate indicator means are known in the art,including fluorescent, radioactive, enzymatic or other ligands, such asavidin/biotin, which are capable of being detected. In preferredembodiments, one may desire to employ a fluorescent label or an enzymetag such as urease, alkaline phosphatase or peroxidase, instead ofradioactive or other environmentally undesirable reagents. In the caseof enzyme tags, colorimetric indicator substrates are known that can beemployed to provide a detection means that is visibly orspectrophotometrically detectable, to identify specific hybridizationwith complementary nucleic acid containing samples.

In general, it is envisioned that the probes or primers described hereinwill be useful as reagents in solution hybridization, as in PCRTM, fordetection of expression of corresponding genes, as well as inembodiments employing a solid phase. In embodiments involving a solidphase, the test DNA (or RNA) is adsorbed or otherwise affixed to aselected matrix or surface. This fixed, single-stranded nucleic acid isthen subjected to hybridization with selected probes under desiredconditions. The conditions selected will depend on the particularcircumstances (depending, for example, on the G+C content, type oftarget nucleic acid, source of nucleic acid, size of hybridizationprobe, etc.). Optimization of hybridization conditions for theparticular application of interest is well known to those of skill inthe art. After washing of the hybridized molecules to removenon-specifically bound probe molecules, hybridization is detected,and/or quantified, by determining the amount of bound label.Representative solid phase hybridization methods are disclosed in U.S.Pat. Nos. 5,843,663, 5,900,481 and 5,919,626. Other methods ofhybridization that may be used in the practice of the present inventionare disclosed in U.S. Pat. Nos. 5,849,481, 5,849,486 and 5,851,772. Therelevant portions of these and other references identified in thissection of the Specification are incorporated herein by reference.

2. Amplification of Nucleic Acids

Nucleic acids used as a template for amplification may be isolated fromcells, tissues or other samples according to standard methodologies(Sambrook et al., 1989). In certain embodiments, analysis is performedon whole cell or tissue homogenates or biological fluid samples withoutsubstantial purification of the template nucleic acid. The nucleic acidmay be genomic DNA or fractionated or whole cell RNA. Where RNA is used,it may be desired to first convert the RNA to a complementary DNA.

The term “primer,” as used herein, is meant to encompass any nucleicacid that is capable of priming the synthesis of a nascent nucleic acidin a template-dependent process. Typically, primers are oligonucleotidesfrom ten to twenty and/or thirty base pairs in length, but longersequences can be employed. Primers may be provided in double-strandedand/or single-stranded form, although the single-stranded form ispreferred.

Pairs of primers designed to selectively hybridize to nucleic acidscorresponding to BoPAGs1-21 or progesterone are contacted with thetemplate nucleic acid under conditions that permit selectivehybridization. Depending upon the desired application, high stringencyhybridization conditions may be selected that will only allowhybridization to sequences that are completely complementary to theprimers. In other embodiments, hybridization may occur under reducedstringency to allow for amplification of nucleic acids contain one ormore mismatches with the primer sequences. Once hybridized, thetemplate-primer complex is contacted with one or more enzymes thatfacilitate template-dependent nucleic acid synthesis. Multiple rounds ofamplification, also referred to as “cycles,” are conducted until asufficient amount of amplification product is produced.

The amplification product may be detected or quantified. In certainapplications, the detection may be performed by visual means.Alternatively, the detection may involve indirect identification of theproduct via chemilluminescence, radioactive scintigraphy of incorporatedradiolabel or fluorescent label or even via a system using electricaland/or thermal impulse signals (Affymax technology; Bellus, 1994).

A number of template dependent processes are available to amplify theoligonucleotide sequences present in a given template sample. One of thebest known amplification methods is the polymerase chain reaction(referred to as PCR™) which is described in detail in U.S. Pat. Nos.4,683,195, 4,683,202 and 4,800,159, and in Innis et al., 1988, each ofwhich is incorporated herein by reference in their entirety.

A reverse transcriptase PCR™ amplification procedure may be performed toquantify the amount of mRNA amplified. Methods of reverse transcribingRNA into cDNA are well known (see Sambrook et al., 1989). Alternativemethods for reverse transcription utilize thermostable DNA polymerases.These methods are described in WO 90/07641. Polymerase chain reactionmethodologies are well known in the art. Representative methods ofRT-PCR are described in U.S. Pat. No. 5,882,864.

Another method for amplification is ligase chain reaction (“LCR”),disclosed in European Application No. 320 308, incorporated herein byreference in its entirety. U.S. Pat. No. 4,883,750 describes a methodsimilar to LCR for binding probe pairs to a target sequence. A methodbased on PCR™ and oligonucleotide ligase assay (OLA), disclosed in U.S.Pat. No. 5,912,148, may also be used.

Alternative methods for amplification of target nucleic acid sequencesthat may be used in the practice of the present invention are disclosedin U.S. Pat. Nos. 5,843,650, 5,846,709, 5,846,783, 5,849,546, 5,849,497,5,849,547, 5,858,652, 5,866,366, 5,916,776, 5,922,574, 5,928,905,5,928,906, 5,932,451, 5,935,825, 5,939,291 and 5,942,391, GB ApplicationNo. 2 202 328, and in PCT Application No. PCT/US89/01025, each of whichis incorporated herein by reference in its entirety.

Qbeta Replicase, described in PCT Application No. PCT/US87/00880, mayalso be used as an amplification method in the present invention. Inthis method, a replicative sequence of RNA that has a regioncomplementary to that of a target is added to a sample in the presenceof an RNA polymerase. The polymerase will copy the replicative sequencewhich may then be detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[alpha-thio]-triphosphates in one strand of arestriction site may also be useful in the amplification of nucleicacids in the present invention (Walker et al., 1992). StrandDisplacement Amplification (SDA), disclosed in U.S. Pat. No. 5,916,779,is another method of carrying out isothermal amplification of nucleicacids which involves multiple rounds of strand displacement andsynthesis, i.e., nick translation.

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS), including nucleic acid sequence basedamplification (NASBA) and 3SR (Kwoh et aL, 1989; Gingeras et al., PCTApplication WO 88/10315, incorporated herein by reference in theirentirety). European Application No. 329 822 disclose a nucleic acidamplification process involving cyclically synthesizing single-strandedRNA (“ssRNA”), ssDNA, and double-stranded DNA (dsDNA), which maybe usedin accordance with the present invention.

PCT Application WO 89/06700 (incorporated herein by reference in itsentirety) disclose a nucleic acid sequence amplification scheme based onthe hybridization of a promoter region/primer sequence to a targetsingle-stranded DNA (“ssDNA”) followed by transcription of many RNAcopies of the sequence. This scheme is not cyclic, i.e., new templatesare not produced from the resultant RNA transcripts. Other amplificationmethods include “race” and “one-sided PCR” (Frobman, 1990; Ohara et al.,1989).

3. Detection of Nucleic Acids

Following any amplification, it may be desirable to separate theamplification product from the template and/or the excess primer. In oneembodiment, amplification products are separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods (Sambrook et al., 1989). Separated amplification products may becut out and eluted from the gel for further manipulation. Using lowmelting point agarose gels, the separated band may be removed by heatingthe gel, followed by extraction of the nucleic acid.

Separation of nucleic acids may also be effected by chromatographictechniques known in art. There are many kinds of chromatography whichmay be used in the practice of the present invention, includingadsorption, partition, ion-exchange, hydroxylapatite, molecular sieve,reverse-phase, column, paper, thin-layer, and gas chromatography as wellas HPLC.

In certain embodiments, the amplification products are visualized. Atypical visualization method involves staining of a gel with ethidiumbromide and visualization of bands, under UV light. Alternatively, ifthe amplification products are integrally labeled with radio- orfluorometrically-labeled nucleotides, the separated amplificationproducts can be exposed to x-ray film or visualized under theappropriate excitatory spectra.

In one embodiment, following separation of amplification products, alabeled nucleic acid probe is brought into contact with the amplifiedmarker sequence. The probe preferably is conjugated to a chromophore butmay be radiolabeled. In another embodiment, the probe is conjugated to abinding partner, such as an antibody or biotin, or another bindingpartner carrying a detectable moiety.

In particular embodiments, detection is by Southern blotting andhybridization with a labeled probe. The techniques involved in Southernblotting are well known to those of skill in the art (see Sambrook etal., 1989). One example of the foregoing is described in U.S. Pat. No.5,279,721, incorporated by reference herein, which discloses anapparatus and method for the automated electrophoresis and transfer ofnucleic acids. The apparatus permits electrophoresis and blottmg withoutexternal manipulation of the gel and is ideally suited to carrying outmethods according to the present invention.

Other methods of nucleic acid detection that may be used in the practiceof the instant invention are disclosed in U.S. Pat. Nos. 5,840,873,5,843,640, 5,843,651, 5,846,708, 5,846,717, 5,846,726, 5,846,729,5,849,487, 5,853,990, 5,853,992, 5,853,993, 5,856,092, 5,861,244,5,863,732, 5,863,753, 5,866,331, 5,905,024, 5,910,407, 5,912,124,5,912,145, 5,919,630, 5,925,517, 5,928,862, 5,928,869, 5,929,227,5,932,413 and 5,935,791, each of which is incorporated herein byreference.

4. Kits

All the essential materials and/or reagents required for detectingBoPAGS1-21 or progesterone in a sample may be assembled together in akit. This generally will comprise a probe or primers designed tohybridize specifically to individual nucleic acids of interest in thepractice of the present invention. Also included may be enzymes suitablefor amplifying nucleic acids, including various polymerases (reversetranscriptase, Taq, etc.), deoxynucleotides and buffers to provide thenecessary reaction mixture for amplification. Such kits may also includeenzymes and other reagents suitable for detection of specific nucleicacids or amplification products. Such kits generally will comprise, insuitable means, distinct containers for each individual reagent orenzyme as well as for each probe or primer pair.

V. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Study #1: Design and Results

Due to high levels of false positive results obtained using a PAG assayonly, a new assay format analyzing both progesterone and PAG levels wasdesigned. A series of designated PAG and progesterone cutoff levels forpregnancy determination based on given concentrations of PAGs andprogesterone were formulated for analysis as set forth below: Day 16  5ng/ml PAG + 3 ng Progesterone/ml Day 16 10 ng/ml PAG + 3 ngProgesterone/ml Day 20  5 ng/ml PAG + 3 ng Progesterone/ml Day 20 10ng/ml PAG + 3 ng Progesterone/ml Day 25 10 ng/ml PAG + 2 ngProgesterone/ml Day 28 10 ng/ml PAG + 2 ng Progesterone/ml Day 30 10ng/ml PAG + 2 ng Progesterone/ml

Assays were carried out as described below. The results of the assaysare set forth below in Table 2. As can be seen, the new assay formatshad a markedly higher accuracy or pregnancy detection at day 25 relativeto progesterone or PAG analysis alone. TABLE 2 Results of BovinePregnancy Test Evaluation Study #1 No. of cows N. of cows Cut off Cutoff false false Day of Pregnancy Range Range for No. of Cows positivenegative Testing for PAG Progesterone Pregnant Open (%) (%) PregnancyRecord — — 54 20 — — Day 45 Palpation — — 53 19 1 (1.4%) 1 (1.4%) PAGAssay Only Day 20 10 ng/ml — 30 44 7 (9.5%) 31 (41.9%) Day 25 10 ng/ml —58 16 6 (8.1%) 2 (2.7%) Day 30 10 ng/ml — 58 16 5 (6.8%) 1 (1.4%)Progesterone Only Day 20 — 3 ng/ml 56 18 5 (6.8%) 3 (4%)   Day 25 — 2ng/ml 59 15 5 (6.8%) 0 (0%)   Day 30 — 2 ng/ml 61 13 9 (12.2%) 2 (2.7%)PAG & Progesterone Day 16  5 ng/ml 3 ng/ml 40 34 6 (8.1%) 20 (27%)   Day20  5 ng/ml 3 ng/ml 42 32 4 (5.4%) 16 (21.6%) Day 20 10 ng/ml 3 ng/ml 2747 3 (4.0%) 30 (41%)   Day 20 10 ng/ml 2 ng/ml 28 46 3 (4.0%) 29 (39%)  Day 25 10 ng/ml 3 ng/ml 51 23 2 (2.7%) 4 (5.4%) Day 25 10 ng/ml 2 ng/ml54 20 2 (2.7%) 2 (2.7%) Day 30 10 ng/ml 3 ng/ml 52 22 2 (2.7%) 5 (6.8%)Day 30 10 ng/ml 2 ng/ml 53 21 2 (2.7%) 3 (4%)  

Example 2 Study #1: Assay Format and Integration of Progesterone Assay

Serum levels of progesterone were obtained using the Coat-a-Count™progesterone kit (Diagnostics Products Corporation, Los Angeles,Calif.). The results of PAG and progesterone serum measurements werecompared with cow pregnancy history beyond day 45 (i.e. pregnancyinformation around day 100) to assess pregnancy diagnosis. As indicatedabove, serum PAG and progesterone assay results and pregnancy datacollected for 74 cows were used for assessing pregnancy diagnosis.

The results are summarized in Table 2. The cutoff range used for PAG was10 ng/ml. This range was selected based on following criteria: a) thepregnancy history of the cow and b) the trend of PAG levels in theserum. If a cow is pregnant, the PAG levels tend to increase from day 16to day 45. A lower cutoff range of 5 ng/ml was also used for day 16 andday 20 because this is very early stage of PAG secretion by theconceptus.

Two cutoff ranges (3 ng/ml and 2 ng/ml) for progesterone were used forProgesterone. The cutoff ranges were selected based on: a) pregnancyhistory of the cows, b) Progesterone levels during estrus cycle andpregnancy in cows. For analysis of false positive results, the cow wasnot pregnant while the test was positive for PAG or Progesterone orPAG+Prog with levels above the indicated cutoff range. For falsenegatives, the cow was pregnant while the test is negative for PAG orProgesterone or PAG+Progesterone with levels below the indicated cutoffrange.

The results were analyzed for PAG only, Progesterone only and thecombination of PAG and Progesterone and compared to pregnancy history.As shown in Table 3, testing at day 25 for either PAG or Progesteroneonly has low false negative results (range from 0% to 2.7%). However,the false positive results (range from 6.8% to 8.1%). The combination ofPAG and progesterone lowers the false positive results to below 3%showing an increased accuracy of pregnancy diagnosis by combining thesetwo measurements. Further, use of the 2 ng cutoff for progesteronerather than 3 ng results in substantially decreased false negatives ondays 25 through 30.

These results establish that a combination of PAG and progesteronemeasurements can be used for detecting pregnancy from day 25 and beyondin cows with low false positive and false negative results. The lowfalse-positive and false-negative results of the combined assay for day25 and beyond also offer a feasibility of developing a reliablepregnancy test for cattle.

In addition, the PAG assay used did not detect any PAG in the serum ofpostpartum cows beyond day 45 after calving (data not shown). This is anadded advantage over the existing PAG1 assay (developed by Sasser et al)since PAG1 remains detectable in day 100 postpartum cows.

The results of the study showed that a combination of PAG andProgesterone measurements allows accurate early detecting of pregnancyin cattle with very low false positive and false negative results. Theassay results showed that the pregnancy status could be detectedsuccessfully by day 25 and beyond in cattle.

Example 3 Sample Collection Study

A sample collection study was performed for evaluating the accuracy ofpregnancy diagnosis using the PAG assay. The study design was to collectserum, milk and urine samples at days 0, 16, 20, 25 and 30 followingartificial insemination of 120 cows. The herd veterinarian palpated thecows at day 45 for pregnancy confirmation. All breeding records andpregnancy data for the cows were also obtained. Data collected fromseveral cows were removed from the study due to re-breeding, pregnancyloss and other problems and not included in the analysis. PAG assayresults for the 74 cows with complete records of breeding and pregnancystatus were used evaluating pregnancy test. The serum samples from these74 cows were used for assaying PAG and progesterone concentration.

Example 4 Polyclonal PAG assay development

The following section describes the polyclonal antibody based assaydevelopment for PAGs. The assay was standardized with an early PAGenriched fraction as antigen and affinity purified rabbit polyclonalantibodies. This assay was used to determine the feasibility ofdetecting PAG during early pregnancy.

Antigen proteins were isolated using day 75-85 placenta. PAGs werefractionated based on their partitioning into acidic, neutral and basicisoelectric pH after binding to pepstatin affinity chromatography. ThePAG eluted from the column at neutral pH were defined as neutral PAG(M3) and the PAG isolated from the column at acidic pH as acidic PAG(M4). Acidic PAG (M4) fraction was used as antigen in the assay.

Antibodies were generated in rabbits according to the standardprotocol-using day 75-85 acidic and neutral PAG. Two rabbits wereimmunized with acidic PAG and two were immunized with neutral PAG inFreunds complete adjuvant. After a two-week interval these rabbits wereboosted with corresponding antigen with incomplete adjuvant. The rabbitswere boosted every two weeks until sufficient antisera were collectedand stored at −70° C. Polyclonal antibodies were affinity purified usingprotein A chromatography and dialyzed in PBS. Purified antibodies werealiquoted and stored at −70° C.

Example 5 Assay Procedure

Two sandwich type immunoassays were developed to evaluate the earlypregnancy by using acidic (M4) and neutral (M3) PAG specific polyclonalantibodies. Immobilized M4 antibodies were reacted with serum samplesand a biotin labeled M4 antibodies were added as secondary antibody.Captured biotin label antibodies were reacted with streptavidin-HRP togenerate a color reaction. Neutral PAG (M3) specific antibodies were notstable in solution and precipitated upon storage. This assay wasdiscontinued after assay reproducibility failed due to the precipitationproblem. The current working assay is the M4 antigen assay, whichcorrespond to early PAG. The calibration range for this assay was from 2to 64 ng/ml. This assay was optimized to obtain the best sensitivity andlow cross reactivity to the non-pregnant serum samples.

Example 6 Study #2: Resynchronization of Dairy Cows and Heifers afterPAG/Progesterone Pregnancy Diagnosis

The inventors designed a second study to test efficacy of a method forrebreeding cows and heifers that are diagnosed as not pregnant after aPAG/progesterone test. Generally, cattle are tested for PAG/progesterone28 to 30 days after breeding and are diagnosed pregnant or non-pregnant.The resynchronization method is implemented on non-pregnant cows 0 to 2days after the PAG/progesterone test. Animals are treated in thefollowing sequence: (i) inject prostaglandin F_(2α) (PGF_(2α); a hormonecausing regression of the corpus luteum); (ii) wait two days, injectgonadotropin releasing hormone (GnRH; a hormone causing ovulation);(iii) wait 0 to 8 hours, (iv) inseminate artificially.

Methods. Dairy cows and heifers were tested for PAG 28 to 30 days afterfirst AI. Cattle diagnosed not pregnant were treated with 5 mL Lutalyse(25 mg PGF_(2α)), two days later were treated with 2 mL Cystorelin (100μg GnRH), and were inseminated 0 to 8 hours after GnRH. Theresynchronization treatment was administered 0 to 2 days after the PAGtest (30 days after first insemination). Pregnancy was determined 30 to60 days after insemination.

Results. Table 3 shows the conception rate for cows and heifers. Dataare separated according to concentrations of progesterone. Two ng/mL isthe cut-off for PAG/Progesterone testing. A cow or heifer with greaterthan 2 ng/mL progesterone would be predicted to have a corpus luteumthat will respond to PGF_(2α) (more-likely to become pregnant afterresynchronization). A cow or heifer with progesterone less than 2 ng/mLwould be predicted to have a corpus luteum that may not respond toPGF_(2α) (less-likely to become pregnant after resynchronization). TABLE3 Conception Rate Conception rate Location Type P4 < 2 ng/mL P4 ≧ 2ng/mL Total Foremost (UMC) Cow 2/15 (13) 9/20 (45) 11/35 (31) Foremost(UMC) Heifer  3/3 (100)  2/4 (50)  5/7 (71) Private dairy Cow  1/3 (33)4/12 (33)  5/15 (33) ALL Locations 6/21 (29) 15/36 (42)  21/57 (37)Conception rate is defined as “no. pregnant/no. inseminated (%)” forcows and heifers that were PAG negative (nonpregnant) with progesterone(P4) either less than or greater than 2 ng/mL.

Discussion. The resynchronization method yielded conception rates thatwere similar to first insemination. Thus, the method appears to be asound approach for handling cows that are not pregnant after firstinsemination.

Example 7 Study #3: Measurement of PAG and Progesterone Levels forAccurate Pregnancy Diagnosis in Cattle

In study #1, the inventors presented data from 79 cows showing that acombination of PAG values above or equal to 10 ng/ml, and progesteronevalues above or equal to 2 ng/ml, would predict pregnancy status of cowswith 97% sensitivity and 97% specificity from 25 days following AI. Inthis follow-up study, the inventors used 270 cows to evaluate combinedtesting for PAG and progesterone for pregnancy diagnosis, as compared toultrasound and palpation results at day 30 and 45 post AI.

Study description and design. The objective of the study was to evaluatethe accuracy, sensitivity (ability to detect pregnant cows) andspecificity (ability to detect open cows) of PAG and progesterone assaysin determining pregnancy status in dairy cows. The study was conductedin 3 commercial dairies. About 300 cows were available at the start ofthe study. The study began on day 0 (day of insemination). Blood sampleswere collected daily from day 20 to 30 days post-insemination and againon day 45 (palpation day). Pregnancy status of the cows was determinedon days 25-29 by transrectal ultrasonography. A second pregnancy exam byrectal palpation was performed at day 45. A small number of cows wereremoved from the study due to health problems. Samples from 270 cowswere available at the end of the study. Serum PAG levels were determinedby PAG ELISA with M4 antiserum. The progesterone levels were measured byusing a commercially available radioimrnunoassay kit.

Results. The results of the analysis are shown in Tables 4 and 5. As instudy #1, the inventors used several cut-off ranges to assess pregnancystatus and, instead of presenting the data as percentages offalse-positives and false-negatives diagnosed, the percentages ofsensitivity and specificity of pregnancy diagnosis are presented.

Table 4 shows the results for PAG, progesterone, PAG and progesteronetests with observed sensitivity and specificity compared to pregnancystatus of the cows determined by ultrasonography and palpation. Thesedata show that combined PAG and P4 test increased the specificity(ability to identify open cows or reducing the false-positives) by morethan 20% in every cut-off ranges examined. In addition, these data alsosupport the claim that PAG and progesterone combined test will increasethe accuracy of pregnancy detection.

In Table 5, the highlighted cut-off values give a minimum of 94%sensitivity and 90% specificity. Again, as in study #1, the inventorsused several cut-off ranges to assess pregnancy status and thepercentages of sensitivity and specificity of pregnancy diagnosis arepresented. The present data shows that it would able to identify 96% ofpregnant cows (i.e., 4% false-negatives) and 91.2% of open cows (8.8%false-positives) on day 25 with 10 ng/ml PAG and 2 ng/ml P4 cut-offrange. The data also shows that increasing the PAG cut-off range. up to26 ng/ml and P4 cut-off at 2 ng/ml consistently improved the specificityof the test (reducing the false-positive diagnosis).

The variability in the PAG cut-off ranges may be due to different set ofreagents (assay standard, dilution serum used for standard) used for PAGELISA. In spite of using the same batch of PAG antibody and PAG antibodyconjugate reagents, a change in the standard curve linearity was notedin this study. This would have influenced the absolute values of PAGmeasured in the serum samples. The progesterone radioimmunoassay used inthe study was an identical commercial kit used in study #1 and no shiftin the standard curve linearity was noted in this assay. TABLE 4 On days25, 27, 29 and 45, a cut-off value for PAG and progesterone wereindividually selected. The values chosen forced sensitivity as close to98% as possible and then the corresponding specificity was determined.The selected cut-offs for the individual days were then combined(right-hand side) to determine how the test might be improved using thetwo analytes. Note however, the values change each test day. IndividualTests Combined PAG + Prog Cut-off Sensitivity Specificity Cut-offSensitivity Specificity Sample Day Model (ng/ml) (%) (%) (ng/ml) (%) (%)25 PAG 6.7 98.0 76.0 (n = 270) Prog 3.8 95.0 72.5  6.7-3.8 92.9 91.8 3.498.0 70.0  6.7-3.4 96.0 91.8 3.0 100.0 66.7  6.7-3.0 97.0 90.6 2.0 100.059.9  6.7-2.0 98.0 89.5 1.0 100.0 49.9  6.7-1.0 98.0 88.3 27 PAG 15.098.0 82.4 (n = 263) Prog 3.9 95.0 71.7 15.0-3.9 92.9 93.3 3.5 98.0 68.115.0-3.5 94.9 92.1 2.7 100.0 61.4 15.0-2.7 98.0 91.5 2.0 100.0 54.215.0-2.0 98.0 90.3 1.0 100.0 38.0 15.0-1.0 98.0 87.3 29 PAG 28.4 98.091.4 (n = 261) Prog 3.8 95.0 64.3 28.4-3.8 92.9 94.5 3.5 98.0 60.728.4-3.5 94.9 94.5 3.1 100.0 52.7 28.4-3.1 98.0 94.5 2.0 100.0 40.528.4-2.0 98.0 93.2 1.0 100.0 30.1 28.4-1.0 98.0 93.2 45 (n = 267) PAG9.9 97.9 88.8 Prog 4.5 95.0 70.2  9.9-4.5 93.8 98.2 4.3 98.0 69.3 9.9-4.3 96.9 98.2 3.5 100.0 57.0  9.9-3.5 97.9 96.5 2.0 100.0 43.3 9.9-2.0 97.9 94.7 1.0 100.0 34.2  9.9-1.0 97.9 93.5

TABLE 5 A set cut-off value for PAG was evaluated over days 25 to 29 todetermine how flexible the test might be. The selected PAG value wasthen combined with various cut-off for progesterone to determine if testspecificity could be adequately improved. Note, the values are constantacross all test days. (Highlighted cut-offs give a minimum of 94%Sensitivity and 90% specificiity on at least days 27, 28 and 29 postAI.) Sensitivity Specificity PAGs Prog. Day Day Day Day Day Day (ng/ml)(ng/ml) 25 27 29 25 27 29 10 0.0 96.0 100.0 100.0 78.9 76.4 80.4 10 1.096.0 100.0 100.0 90.1 83.6 85.3 10 2.0 96.0 100.0 100.0 91.2 86.7 85.910 3.0 94.9 99.0 100.0 92.4 87.9 89.0 10 4.0 88.9 93.9 94.9 94.2 90.391.4 12 0.0 92.9 99.0 100.0 80.1 78.2 81.6 12 1.0 92.9 99.0 100.0 90.184.8 85.9 12 2.0 92.9 99.0 100.0 91.2 87.9 86.5 12 3.0 91.9 98.0 100.092.4 89.1 89.6 12 4.0 85.9 92.9 94.9 94.2 91.5 91.4 14 0.0 89.9 99.099.0 82.5 81.2 84.7 14 1.0 89.9 99.0 99.0 91.2 86.7 87.7 14 2.0 89.999.0 99.0 91.8 89.7 87.7 14 3.0 88.9 98.0 99.0 93.0 90.9 90.8 14 4.082.8 92.9 93.9 94.7 93.3 92.6 16 0.0 85.9 98.0 99.0 85.4 84.2 85.3 161.0 85.9 98.0 99.0 91.8 88.5 87.7 16 2.0 85.9 98.0 99.0 92.4 90.9 87.716 3.0 84.8 96.9 99.0 93.6 92.1 90.8 16 4.0 78.8 91.8 93.9 95.3 93.992.6 18 0.0 80.8 98.0 99.0 86.5 85.5 85.9 18 1.0 80.8 98.0 99.0 93.089.1 88.3 18 2.0 80.8 98.0 99.0 93.6 91.5 88.3 18 3.0 79.8 96.9 99.094.2 92.7 91.4 18 4.0 73.7 91.8 93.9 95.9 94.5 93.3 20 0.0 77.8 98.099.0 88.3 86.7 89.0 20 1.0 77.8 98.0 99.0 94.2 90.3 91.4 20 2.0 77.898.0 99.0 94.2 92.1 91.4 20 3.0 76.8 96.9 99.0 94.2 92.7 93.3 20 4.071.7 91.8 93.9 95.9 94.5 93.9 22 0.0 76.8 98.0 99.0 88.9 86.7 89.6 221.0 76.8 98.0 99.0 94.7 90.9 92.0 22 2.0 76.8 98.0 99.0 94.7 92.1 92.022 3.0 75.8 96.9 99.0 94.7 92.7 93.3 22 4.0 70.7 91.8 93.9 95.9 94.593.9 24 0.0 74.7 98.0 99.0 90.6 87.3 89.6 24 1.0 74.7 98.0 99.0 95.390.3 92.0 24 2.0 74.7 98.0 99.0 95.3 92.7 92.0 24 3.0 73.7 96.9 99.095.3 93.3 93.3 24 4.0 68.7 91.8 93.9 95.9 94.5 93.9 26 0.0 72.7 95.999.0 91.8 88.5 90.8 26 1.0 72.7 95.9 99.0 95.9 92.1 93.3 26 2.0 72.795.9 99.0 95.9 93.3 93.3 26 3.0 71.7 94.9 99.0 95.9 93.9 94.5 26 4.066.7 89.8 93.9 95.9 95.2 94.5

In summary, the results of study #3 support the utility of the claimedinvention. Combined testing of PAG and progesterone considerably reducesthe false-positive and false-negative results and improves the accuracyof pregnancy diagnosis in cows.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and in the steps or in the sequence of steps of the methoddescribed herein without departing from the concept, spirit and scope ofthe invention. More specifically, it will be apparent that certainagents which are both chemically and physiologically related may besubstituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference:

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1. A method for the early detection of pregnancy in a bovine animalcomprising: (a) obtaining a sample from said bovine animal; (b)measuring the level of at least one bovine pregnancy associatedglycoprotein (BOPAG) in said sample; and (c) measuring the level ofprogesterone in said sample, wherein elevated levels of BoPAG andprogesterone indicate that said bovine animal is pregnant.
 2. The methodof claim 1, wherein said sample is saliva, serum, blood, milk or urine.3-7. (canceled)
 8. The method of claim 1, wherein said sample isobtained from said animal at days 16 to 30 post-insemination.
 9. Themethod of claim 8, wherein said sample is obtained from said animal atday 20, 21, 22, 23, 24, 25, 26, 27 or 28 post-insemination.
 10. Themethod of claim 1, further comprising measuring the level of more thatone BoPAG.
 11. The method of claim 1, wherein said BoPAG is selectedfrom the group consisting of BoPAG1, BoPAG2, BoPAG3, BoPAG4, BoPAG5,BoPAG6, BoPAG7, BoPAG8, BoPAG9, BoPAG7v; BoPAG9v; BoPAG10, BoPAG11,BoPAG12, BoPAG13, BoPAG14, BoPAG15; BoPAG16; BoPAG17; BoPAG18; BoPAG19;BoPAG20 or BoPAG21.
 12. The method of claim 1, wherein said BoPAG ispresent in early pregnancy.
 13. The method of claim 12, wherein saidBoPAG is selected from the group consisting of BoPAG2, BoPAG4, BoPAG5,BoPAG6, BoPAG7, BoPAG8, BoPAG9, BoPAG10, BoPAG11 and BoPAG21.
 14. Themethod of claim 1, wherein said BoPAG is present throughout pregnancy.15. The method of claim 14, wherein said BoPAG is selected from thegroup consisting of BoPAG2.
 16. The method of claim 1, wherein saidBoPAG is present in early pregnancy and absent at about two monthspost-partum.
 17. The method of claim 16, wherein said BoPAG is selectedfrom the group consisting of BoPAG4, BoPAG5, BoPAG6, BoPAG7, BoPAG9,BoPAG11 and BoPAG21.
 18. The method of claim 1, wherein said measuringBoPAG levels comprises immunologic detection.
 19. The method of claim18, wherein said immunologic detection comprises detecting a pluralityof BoPAGs with polyclonal antisera.
 20. The method of claim 19, whereinsaid polyclonal antisera lack substantial binding activity to BoPAG1.21. The method of claim 19, wherein said polyclonal antisera is preparedagainst acidic fraction of day 60-85 BoPAG.
 22. The method of claim 19,wherein said polyclonal antisera is prepared against neutral fraction ofday 60-85 BoPAG.
 23. The method of claim 18, wherein said immunologicdetection comprises detecting a single BoPAG with a monoclonal antibodypreparation.
 24. The method of claim 18, wherein said immunologicdetection comprises detection of multiple BoPAGs with a monoclonalantibody preparation.
 25. The method of claim 18, wherein saidimmunologic detection comprises a method selected from the groupconsisting of ELISA, RIA and Western blot. 26-27. (canceled)
 28. Themethod of claim 25, wherein said ELISA is a sandwich ELISA comprisingbinding of a BoPAG to a first antibody preparation fixed to a substrateand a second antibody preparation labeled with an enzyme.
 29. The methodof claim 28, wherein said enzyme is alkaline phosphatase or horseradishperoxidase.
 30. The method claim 4, wherein elevated level of totalBoPAG is from about 5 to about 10 ng/ml of serum.
 31. The method ofclaim 30, wherein elevated level of total BoPAG is about 5 ng/ml. 32.The method of claim 30, wherein elevated level of total BoPAG is about10 ng/ml.
 33. The method of claim 1, wherein measuring BoPAG levelscomprises nucleic acid hybridization.
 34. The method of claim 33,wherein nucleic acid hybridization comprises a method selected from thegroup consisting of Northern blotting amplification and RT-PCR. 35-36.(canceled)
 37. The method of claim 1, wherein measuring progesteronelevels comprises immunologic detection.
 38. The method of claim 37,wherein said immunologic detection comprises detecting progesterone withpolyclonal antisera.
 39. The method of claim 37, wherein saidimmunologic detection comprises detecting progesterone with a monoclonalantibody preparation.
 40. The method of claim 37, wherein saidimmunologic detection comprises a method selected from the groupconsisting of ELISA, RIA and Western blot. 41-42. (canceled)
 43. Themethod of claim 40, wherein said ELISA is a sandwich ELISA comprisingbinding of progesterone to a first antibody preparation fixed to asubstrate and a second antibody preparation labeled with an enzyme. 44.The method of claim 43, wherein said enzyme is alkaline phosphatase orhorseradish peroxidase.
 45. The method claim 4, wherein elevated levelof progesterone is about 2 ng/ml of serum.
 46. The method of claim 1,wherein measuring progesterone levels comprises measuring progesteronebiosynthesis pathway enzyme levels by nucleic acid hybridization,immunologic detection or enzyme activity measurement.
 47. The method ofclaim 46, wherein nucleic acid hybridization comprises a method selectedfrom the group consisting of Northern blotting, amplification andRT-PCR. 48-49. (canceled)
 50. The method of claim 4, wherein said sampleis obtained at day 25 post-insemination, and the elevated levels ofBoPAG and progesterone are 10 ng/ml and 2 ng/ml, respectively.
 51. Themethod of claim 1, further comprising a positive control sample from apregnant bovine animal.
 52. The method of claim 1, further comprising anegative control sample from a non-pregnant bovine animal.
 53. Themethod of claim 1, further comprising measuring BoPAG and progesteronelevels from a second sample from said bovine animal at a second point intime.
 54. A method of making a breeding decision for a bovine animalcomprising: (a) obtaining a sample from said bovine animal, wherein saidbovine animal is suspected of being pregnant; (b) measuring the level ofat least one bovine pregnancy associated antigen (BoPAG) in said sample;and (c) measuring the level of progesterone in said sample, wherein: (i)elevated levels of BoPAG and progesterone indicate that said bovineanimal is pregnant, and no further steps need be taken; (ii)non-elevated levels of BoPAG and progesterone indicate that said bovineanimal is not pregnant, and should be injected withgonadotropin-releasing hormone (GnRH), and about seven days later,injected with prostaglandin F₂, (PGF), followed by re-insemination;(iii) elevated levels of BoPAG and non-elevated levels of progesteroneindicate that said bovine animal is not pregnant due to early embryodeath and should be injected with GnRH, and about seven days later,injected with PGF, followed by re-insemination; or (iv) non-elevatedlevels of BoPAG and elevated levels of progesterone indicate that saidbovine animal is not pregnant, and should be injected with PGF, followedby re-insemination.
 55. The method of claim 54, further comprising insteps (ii), (iii) and (iv), about 48 hours after PGF injection andbefore re-insemination, administering a second injection of GnRH. 56.The method of claim 54, further comprising, prior to step (a),inseminating said bovine animal.
 57. The method of claim 54, whereinsaid PGF injection is administered at day 28 post-insemination andwherein said re-insemination is carried out at day 31 post-insemination.